Proc. Nat. Acad. Sci. USA Vol. 73, No. 2, pp. 386-390, February 1976 Biochemistry

Evidence that acyl coenzyme A synthetase activity is required for repression of yeast acetyl coenzyme A carboxylase by exogenous fatty acids (Saccharomyces cerevisiae/mutant/cerulenin/regulation of fatty acid synthesis) TATSUYUKI KAMIRYO, SAMPATH PARTHASARATHY, AND SHOSAKU NUMA Department of Medical Chemistry, Kyoto University Faculty of Medicine, Kyoto 606, Japan Communicated by Feodor Lynen, November 17,1975

ABSTRACT The cellular content of acetyl-CoA carboxyl- is the immediate mediator of the repressive effect. A likely ase [acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC candidate for such a mediator would be either fatty acid it- 6.4.1.21 in Saccharomyces cerevisiae is reduced by the addi- tion of long-chain fatty acids to the culture medium. Mutant self or some compound metabolically derived from it. strains of S. cerevisiae defective in acyl-CoA synthetase In the present investigation, mutant strains of S. cerevisiae [acid:CoA ligase (AMP-forming), EC 6.2.1.31 were isolated defective in acyl-CoA synthetase [acid:CoA ligase (AMP- and used to determine whether fatty acid itself or a metabo- forming), EC 6.2.1.3] were isolated and utilized to deter- lite of fatty acid is more directly responsible for the repres- mine whether fatty acid itself or a metabolite of fatty acid is sion of acetyl-CoA carboxylase. Cells of the mutant strains more directly responsible for the repression of acetyl-CoA were capable of incorporating fatty acid to an extent compa- rable to that observed with the wild-type strain, but they ac- carboxylase observed upon addition of fatty acid to the cul- cumulated markedly more of the incororated fatty acid in ture medium. Such mutants promised to be useful for this the nonesterified form than did the wild-type cells. The level purpose, since acyl-CoA can be regarded as an obligatory in- of acetyl-CoA carboxylase activity in the mutants, in contrast termediate for further metabolism of fatty acid. Studies with to that in the wild-type strain, was hardly affected by the ad- these mutants have provided evidence indicating that the re- dition of fatty acids to the medium. These results indicate pression of acetyl-CoA carboxylase by exogenous fatty acid that the activation of exogenous fatty acid is required for the repression of acetyl-CoA carboxylase, supporting the view is mediated by some metabolite of fatty acid rather than by that the repressive effect is mediated by some compound me- nonesterified fatty acid. tabolically derived from fatty acid. The synthesis de novo of long-chain fatty acids in yeast pro- MATERIALS AND METHODS ceeds via malonyl-CoA, which is the product of the reaction Chemicals. Fatty acids were purchased from Nakarai catalyzed by acetyl-CoA carboxylase [acetyl-CoA:carbon- (Kyoto, Japan). [9,10-3H]Palmitic acid was obtained from dioxide ligase (ADP-forming), EC 6.4.1.2] (1). This enzyme the Commissariat a l'Energie Atomique (Saclay, France), plays a critical role in the regulation of fatty acid synthesis and [U-'4C]palmitic acid and sodium [1-'4C]acetate from in yeast (2) as well as in animal tissues (3). Our previous the Radiochemical Centre (Amersham, England). Phospha- studies have demonstrated that the level of acetyl-CoA car- tidylcholine, phosphatidylethanolamine, and triglyceride boxylase activity in Saccharomyces cerevisiae is lowered by were products of Serdary (London, Canada). 1,2-Diglycer- the addition of long-chain fatty acids to the culture medium ide was prepared as described previously (6). CoA and ATP- (2). The results of immunochemical titrations have indicated were obtained from Boehringer (Mannheim, Germany), and that this decrease in the activity level is due-to a reduced cel- Triton X-100 from Rohm and Haas (Philadelphia, Pa.). Sili- lular content of the enzyme rather than to an altered catalyt- ca gel G plates for thin-layer chromatography were pur- ic efficiency of individual enzyme molecules. Moreover, the chased from Merck (Rahway, N.J.). Cerulenin was a gener- extent of the decrease in the cellular content of the enzyme ous gift of Dr. S. Omura and Dr. J. Awaya (Kitasato Univer- can account for the reduced rate of [14C]acetate incorpora- sity, Tokyo, Japan). tion into fatty acids by cells grown with exogenous fatty Yeast Strains and Culture Media. A haploid yeast, Sac- acid. charomyces cerevtsiae X 2180-1B, a mating type, was used An analogous effect of exogenous fatty acids on the cellu- as a wild-type strain. Mutant strains B-53 and B-201 with lar content of acetyl-CoA carboxylase has been shown with defective acyl-CoA synthetase were isolated from the wild- human skin fibroblasts (4) and rat hepatocytes (5) in culture. type strain as described below. YPS-medium used for ctil- Furthermore, isotopic leucine incorporation studies with the ture of yeast cells consisted of 0.3% Bacto-yeast extract use of immunochemical techniques have revealed that the (Difco, Detroit, Mich.), 0.5% Bacto-peptone (Difco), 0.5% reduction of the enzyme content upon addition of fatty acid KH2PO4, 0.5% K2HPO4, and 2% sucrose. YPS-agar con- to the culture medium can be ascribed to a decrease in the tained 1.5% Bacto-agar (Difco) in addition to YPS-medium. rate of synthesis of the enzyme, while the rate of degrada- YPSB-medium used to examine the effect of added fatty tion of the enzyme is not affected by exogenous fatty acid acids on the level of acetyl-CoA carboxylase activity was (5). On the basis of these findings, it appears reasonable to composed of 0.7% Bacto-yeast extract, 0.7% Bacto-peptone, assume that the observed decrease in the acetyl-CoA carbox- 0.5% KH2PO4, 0.5% K2HPO4, 1% sucrose, and 1% Brij 58 ylase content of S. cerevisiae likewise represents a repression (Kao-Atlas, Tokyo, Japan). of the enzyme. In order to understand the mechanisms Isolation of Mutants. X 2180-1B cells grown in YPS- underlying the repression of acetyl-CoA carboxylase by ex- medium at 240 were mutagenized with 2.5% ethylmeth- ogenous fatty acid, it is of crucial importance to know what anesulfonate (Nakarai) according to the procedure described 386 Downloaded by guest on September 27, 2021 Biochemistry: Kamiryo et al. Proc. Nat. Acad. Sci. USA 73 (1976) 387 by Fink (7) to a survival rate of 20-30%. After growth in Louis, Mo.) as the standard. YPS-medium at 24° for 40-60 hr, the mutagenized cells [14CJPalmitate or [14C]Acetate Incorporation Studies. A were transferred to YPS-medium containing 0.17 mM culture grown in YPS-medium at 240 overnight was inocu- [9,10-3H]palmitic acid (1.6 Ci/mmol), 0.3% Brij 58, and 25 lated in 5 ml of YPS-medium to give a density of approxi- ,MM cerulenin, and were incubated at 370 for 6 hr. The cells mately 3 X 106 cells per ml. After incubation at 36° for 2 hr were washed three times with S-solution (0.1 M NaCi and 10 with agitation, 0.5 MCi of [U-'4C]palmitic acid (2 mCi/ mM Mg9l2) containing 0.3% Brij 58 and then twice with S- mmol) dissolved in 10-50 MAl of ethanol or 5 MCi of sodium solution. The washed cells, suspended in S-solution to give a [1-'4C]acetate (0.1 mCi/mmol) was added. After further in- density of about 10P cells per ml, were stored at 40 for 71 cubation at 360 for 2 hr, the cells were heated in boiling days. After the storage, the cells were plated on YPS-agar water for 2 min and collected by centrifugation. They were and incubated at 240. The survival rate during the storage then washed three times at room temperature either with 3 was 2 X 10-5. The colonies arising from the surviving cells ml of S-solution containing 1% Brij 58 and 0.5 mM nonra- were replica-plated onto YPS-agar containing 25 MAM ceru- dioactive palmitic acid ([14C]palmitate-labeled cells) or with lenin, 0.3 mM palmitic acid, and 0.3% Brij 58, and were in- 3 ml of S-solution ([14C]acetate-labeled cells). The washed cubated at 370 for 5 days. Strains which failed to grow on cells were suspended in S-solution. A portion of the cell sus- this medium were isolated from the master plates. After sin- pension was used to determine the uptake of the radioactive gle colony isolation, they were tested for a mating type. The precursor by the cells with the use of the scintillator solution strains with the desired phenotype were grown in 5 ml of of Patterson and Greene (12). The uptake proceeded almost YPS-medium at 240 overnight and were treated with tolu- linearly for 2 hr. To another portion of the cell suspension ene according to the method of Serrano et al. (8) at a density was added 0.3 g (wet weight) of boiled carrier cells, and the of 1 to 2 X 108 cells per ml. The toluenized cells were cells were collected by centrifugation and ground with 1 g washed with 3 ml of 0.1 M potassium phosphate buffer (pH of levigated alumina (Wako, Osaka, Japan), which has little, 6.5) containing 5 mM 2-mercaptoethanol and 1 mM EDTA. if any, interaction with lipids. Lipids were extracted from The washed cells were suspended in 0.5 ml of the same solu- the disrupted cells with 2.5 ml of chloroform-methanol 2:1. tion and assayed for acyl-CoA synthetase activity by the hy- Extraction was repeated two more times with 1.5 ml of the droxamic acid method (see below). Roughly 4% of the survi- same solvent. The combined extracts were washed succes- vors from the radiation suicide exhibited low acyl-CoA syn- sively with 0.9 ml of 0.1 M HC1 and with 2 ml of methanol- thetase activity at 370. 0.2 M HC1 1:1, dried under reduced pressure, and dissolved Assays of Acyl-CoA Synthetase and Acetyl-CoA Carbox- in 0.5 ml of chloroform-methanol 2:1. The recovery of ra- ylase. Acyl-CoA synthetase activity was assayed either by dioactivity was more than 90%. Lipids were analyzed by the hydroxamic acid method or by the isotopic method. For thin-layer chromatography on a silica gel G plate with either the screening of mutants, the toluenized cells were tested for n-hexane-diethyl ether-acetic acid 30:6:0.5 or benzene-di- the enzyme activity at 370 by the hydroxamic acid method ethyl ether-ethanol-acetic acid 50:40:2:0.2 as the developing as described by Pande and Mead (9) with the use of potassi- solvent. Lipids were visualized with iodine vapor and identi- um oleate as the substrate. The enzyme activity of the par- fied by comparison with authentic samples. Silica gel zones ticulate preparations was determined by the isotopic method containing radioactive lipids were scraped into vials and according to the procedure of Banis and Tove (10) with counted in the scintillator solution mentioned above. Radio- some modifications. For this purpose, the yeast homogenate, gas-liquid chromatography of methyl esters of fatty acids which was prepared with a cell homogenizer (Braun, Mel- was carried out under the conditions described previously sungen, Germany) as described previously (2), was centri- (13). fuged at 20,200 X g and 40 for 15 min. The supernatant was further centrifuged at 105,000 X g and 40 for 1 hr. The re- RESULTS sulting pellet was washed once with ice-cold 0.1 M potassi- Acyl-CoA Synthetase Activity of Mutants. Mutants of S. um phosphate buffer (pH 6.5) containing 5 mM 2-mercapto- cerevisiae with defective acyl-CoA synthetase would be via- ethanol and 1 mM EDTA, and was used for the assay. The ble, since the final products formed in vitro by the fatty acid reaction mixture contained 40,Mmol of Tris-HCI buffer (pH synthetase complex from this organism have been shown to 7.5), 4 Mmol of L-cysteine, 4 Mmol of KF, 3 Mumol of MgCl2, be long-chain acyl-CoA thioesters rather than nonesterified 1.5 Mumol of ATP, 0.1 Mumol of CoA, 0.2 Mmol of potassium fatty acids (14). However, the action of acyl-CoA synthetase [U-'4C]palmitate (0.25 mCi/mmol), 0.1 mg of Triton X-100, would become indispensable when yeast cells in which fatty and 50-200 ,ug of enzyme in a total volume of 0.2 ml. Prior acid synthesis de novo is blocked are grown in a medium to the assay, the enzyme was preincubated for 10 min in a supplemented with fatty acid. Cerulenin, an antifungal anti- mixture (0.19 ml) containing all the components of the reac- biotic produced by Cephalosporium caerulens (15), is tion mixture except CoA. The reaction was then initiated by known to block fatty acid synthesis by inhibiting specifically the addition of CoA (10 Ml) and terminated after 20 min ,B-ketoacyl-(acyl-carrier-protein) synthase [acyl-(acyl-car- with 2.5 ml of isopropanol-n-heptane-1 M H2SO4 40:10:1. rier-protein):malonyl-(acyl-carrier-protein) C-acyltransfer- The temperature for the preincubation as well as for the re- ase (decarboxylating), EC 2.3.1.41] (16). Accordingly, it was action was 360 unless otherwise stated. Under the conditions expected that mutants defective in acyl-CoA synthetase used, the reaction proceeded linearly for at least 20 min. All would not survive in a medium containing cerulenin and the determinations were carried out in the range where the fatty acid, while they would grow in a normal sucrose medi- initial reaction velocity was proportional to the amount of um. In contrast, the wild-type cells would grow in both enzyme added. Essentially no reaction occurred in the ab- media. On the basis of this rationale, mutant strains B-53 sence of added CoA. Acetyl-CoA carboxylase activity was and B-201 were isolated as described in Materials and assayed at 360 by the H14CO,3-_fixation method as described Methods. previously (2). Protein was determined by the method of As shown in Table 1, the particulate preparations derived Lowry et al. (1 1) with bovine serum albumin (Sigma, St. from mutants B-53 and B-201 exhibited very low levels of Downloaded by guest on September 27, 2021 388 Biochemistry: Kamiryo et al. Proc. Nat. Acad. Sci. USA 73 (1976)

Table 1. Acyl-CoA synthetase activity of wild-type It was expected that in the acyl-CoA synthetase mutants and mutant yeast the [14C]palmitate incorporated would exist as nonesterified fatty acid rather than in esterified forms. Lipids were ex- Specific activity tracted from the washed labeled cells (more than 90% of the (nmol/min per mg of protein) radioactivity was recovered), and the distribution of radioac- tivity in various lipid species was examined by thin-layer Strain Exp. 1 Exp. 2 chromatography. The results presented in Table 2 revealed X 2180-1B 50.8 45.8 that the mutant cells actually accumulated markedly more B-201 1.2 1.0 labeled nonesterified fatty acid (30-44%) than did the wild- B-53 2.5 1.4 type cells (7%). The radioactive nonesterified fatty acid de- rived from the mutant and the wild-type cells was eluted Cells were grown at 24° in 500 ml of YPS-medium and har- and analyzed by radio-gas-liquid chromatography after vested during the mid-logarithmic phase. Enzyme assays were methylation. The results demonstrated that, for all three conducted as described in Materials and Methods. strains, palmitic acid was the only fatty acid containing ra- dioactivity. It is uncertain whether the esterification ob- acyl-CoA synthetase activity. Although the acyl-CoA synthe- served in the mutant cells was due to a residual activity of tase activity of mutant B-201 was negligible at 360, 5-10% acyl-CoA synthetase or to some unknown mechanism. of the activity of the wild-type strain was detected when the ['4CJAcetate Incorporation Studies. As mentioned above, assay (including 10-min preincubation) was conducted at the final products formed in vitro by the fatty acid synthe- 240. The activity of mutant B-201 at 240 was completely tase complex from S. cerevisiae are long-chain acyl-CoA abolished by preincubating the enzyme preparation at 400 thioesters rather than nonesterified fatty acids (14). There- for 30 min, while the activity of the wild-type preparation fore, the activity of acyl-CoA synthetase would not be re- was unaffected under the same conditions. These results quired when fatty acids formed by synthesis de novo in cells suggest that strain B-201 has a mutation in the structural are further metabolized. In the experiments shown in Table gene of-acyl-CoA synthetase. 3, cells were incubated with [14C]acetate, and the distribu- [14CJPalmitate Incorporation Studies. In view of the re- tion of radioactivity in various lipid species was determined. port that mutants of Escherichia coli lacking acyl-CoA syn- In contrast to the results of the analogous experiments with thetase fail to take up oleic acid (17), experiments were car- ['4C]palmitate (Table 2), very little labeled nonesterified ried out to examine the uptake of fatty acid by the yeast mu- fatty acid accumulated in either the wild-type or the mutant tants B-53 and B-201. As is evident from Table 2, both mu- cells. These data indicate that, in agreement with the results tants, despite their very low acyl-CoA synthetase activity, of the in vitro studies, the final products of the fatty acid were capable of taking up [14C]palmitate to an extent com- synthetase reaction in vivo are acyl-CoA derivatives. Fur- parable to that observed with the wild-type strain. In these thermore, the data seem to exclude the possibility that the experiments, labeled cells were washed three times with a mutants used might have an abnormally high activity of solution containing a detergent and nonradioactive palmitic acyl-CoA hydrolase (EC 3.1.2.2), which would result in an acid. The radioactivity associated with the washed cells can apparent deficiency of acyl-CoA synthetase. be regarded as having actually been incorporated into the Effect of Exogenous Fatty Acids on Acetyl-CoA Carbox- cells rather than as having merely been adsorbed on the cell ylase of Mutants. As described above, the acetyl-CoA car- surface. This was evidenced by the facts that further wash- boxylase content of the wild-type strain of S. cerevisiae is re- ing of the cells removed only a negligible amount of radio- duced upon addition of fatty acids to the culture medium. If activity and that less than 10% of the radioactivity extracted this repressive phenomenon were mediated by nonesterified from the wild-type cells was found in nonesterified fatty fatty acid, it would be expected to occur in the acyl-CoA acid as described below. synthetase mutants to an extent equal to or greater than that

Table 2. Uptake of ['4C I palmitate by growing cells of wild-type and mutant yeast and distribution of radioactivity in-cellular lipids Distribution of radioactivity (%)t Rate of uptake* Nonesterified Strain (cpm x 10-4/hr) fatty acid4: Polar lipid § Triglyceride Diglyceridel X 2180-1B (7) 35 ± 4 7 ± 1 72 ± 2 12 ± 2 9 ± 1 B-201 (5) 23 ± 2 44 ± 6 45 ± 5 5 ± 1 6 ± 1 B-53(4) 27± 2 30± 2 43± 1 27± 1 Experimental details are described in Materials and Methods. The figures in parentheses after strain refer to the number of experiments, and the results are expressed as means + SEM. When the entire thin-layer plate was automatically scanned, over 90% of the radioactivity was recovered in the areas containing the lipid species listed; the remainder was found at the solvent front and was not included in the cal- culations. * Per unit of optical density at 660 nm of the cell suspension. t The sum of the radioactivities determined is taken as 100%. T The identity was further confirmed by rechromatography on a thin-layer plate with n-hexane-diethyl ether-methanol-acetic acid 90:20:3:2 as well as with n-heptane-diisopropyl ether-acetic acid 60:40:3. § The majority of the radioactivity was located in phosphatidylcholine and phosphatidylethanolamine by thin-layer chromatography with chloroform-methanol-water 65:25:4. For mutant B-53, radioactivity in diglyceride was not determined except in one experiment, in which 4% of the total radioactivity was found in this fraction. Downloaded by guest on September 27, 2021 Biochemistry: Kamiryo et al. Proc. Nat. Acad. Sci. USA 73 (1976) 389

Table 3. Uptake of [14C] acetate by growing cells of Table 5. Effect of cerulenin on the repression of wild-type and mutant yeast and distribution of acetyl-CoA carboxylase in wild-type yeast radioactivity in cellular lipids Specific activity Distribution of radioactivity (nmol/min per mg of protein) (%)t Addition Exp. 1 Exp. 2 Non- Rate of esteri- None (control) 10.0 11.5 uptake* fied Tri- Di- Fatty acids 5.4 (54) 5.9 (51) (cpm X fatty Polar glyc- glyc- Fatty acids Exp. Strain 10-3/hr) acid lipid eride eride and cerulenin 8.8 (88) 10.2 (89)

1 X2180-1B 28 4 67 10 19 Experimental details were as described for Table 4. The concen- tration of cerulenin added was 25 AM. The figures in parentheses B-201 22 5 68 7 19 represent percent of the control values. B-53 21 7 52 22 18 2 X 2180-1B 33 4 67 10 18 B-201 22 5 69 7 19 B-53 20 9 51 22 18 carboxylase by exogenous fatty acids is not very large, the repressed activity level being about 50% of the control value. Experimental details are described in Materials and Methods. This fact suggests the possibility that the enzyme might be Scanning of the entire thin-layer plate showed that at least 86% of repressed to a certain extent even in cells grown without ad- the radioactivity was recovered in the areas containing the lipid species listed; the remainder was present at the solvent front and dition of fatty acid. Experiments were designed, therefore, was not included in the calculations. to examine the repressive effect of the putative fatty acid * Per unit of optical density at 660 nm of the cell suspension. metabolite formed by synthesis de novo in the wild-type t The sum of the radioactivities determined is taken as 100%. cells. In the experiments represented in Table 5, fatty acid synthesis was blocked by the addition of cerulenin at a con- observed in the wild-type strain. The results given in Table 4 centration of 25 MM, which was shown to block [14C]acetate demonstrated, however, that the level of acetyl-CoA carbox- incorporation into fatty acids in S. cerevistae almost com- ylase activity in the mutants was hardly affected by the ad- pletely. The results revealed that acetyl-CoA carboxylase un- dition of fatty acids to the medium, while, as reported pre- derwent only slight (about 10%) repression in the cells grown viously (2), the activity level in the wild-type strain was de- in the presence of cerulenin and fatty acids, while the extent creased to about 50% of the control value. This finding indi- of repression in the cells grown with fatty acids alone was as cates that the activation of exogenous fatty acid is required usual (about 50%). These data suggest that the blockage of for the repression of acetyl-CoA carboxylase, thus support- fatty acid synthesis due to cerulenin results in a decrease in ing the viewsthat the repressive effect is mediated by some the formation of the fatty acid metabolite which is responsi- compound metabolically derived from fatty acid. In the ex- ble for the repression of acetyl-CoA carboxylase. The results periments just mentioned, a mixture of palmitic acid, oleic given in Table 5 could also be accounted for by a possible in- acid, and linoleic acid was used to examine the effect of hibition of acyl-CoA synthetase by cerulenin. This possibility fatty acids. It is to be noted in this context that each of these was excluded, however, by experiments in vitro as well as in fatty acids is an effective substrate for acyl-CoA synthetase Wvo. Cerulenin at concentrations ranging from 15 AM to from S. cerevlsiae (18). 150 MM did not affect the acyl-CoA synthetase activity of Effect of Cerulenin on the Repression of Acetyl-CoA the particulate preparations. In [14C]palmitate incorporation Carboxylase. The extent of the repression of acetyl-CoA studies, the presence of 25 MM cerulenin in the culture me- dium did not alter the distribution of radioactivity between Table 4. Acetyl-CoA carboxylase activity of wild-type nonesterified fatty acid and esterified species. and mutant yeast grown with or without addition of fatty acids DISCUSSION Specific activity (nmol/min per mg of protein) The present investigation has demonstrated that mutant strains of S. cerevisiae defective in acyl-CoA synthetase, in Without fatty contrast to the wild-type strain, exhibit little repression of Exp. Strain acids (control) With fatty acids acetyl-CoA carboxylase when cells are grown in a medium supplemented with long-chain fatty acids. This finding indi- 1 X2180-1B 11.3 6.0 (53) cates that the immediate mediator of the effect is B-201 6.6 5.8 (88) repressive B-53 11.0 9.6 (87) not fatty acid itself but some compound metabolically de- 2 X 2180-1B 11.5 5.9 (51) rived from it. The fatty acid metabolite responsible for the B-201* 8.6 7.7 (90) repression of acetyl-CoA carboxylase might be long-chain B-53 11.2 10.0 (89) acyl-CoA thioester, its elongation product, or some lipid product formed by esterification. Malonyl-CoA, the imme- Cells were grown at 35° in 500 ml of YPSB-medium with or with- diate product of the acetyl-CoA carboxylase reaction, does out addition of a mixture of palmitate, oleate, and linoleate (1 mM not appear to be the mediator of the repressive effect, since each) and were harvested when the optical density at 660 nm of the only slight repression of acetyl-CoA carboxylase is observed culture was approximately 2.5. Enzyme assays were carried out as described in Materials and Methods. The figures in parentheses in the presence of cervlenin, which would increase the intra- represent percent of the control values. cellular concentration of malonyl-CoA by inhibiting fatty * Cells were grown at 33°. acid synthetase. Downloaded by guest on September 27, 2021 390 Biochemistry: Kamiryo et al. Proc. Nat. Acad. Sci. USA 73-(1976)

We are indebted to Dr. S. Omura and Dr. J. Awaya for a kind 8. Serrano, R., Gancedo, J. M. & Gancedo, C. (1973) Eur. J. Bto- gift of cerulenin. We thank also Dr. S. C. Hubbard for critically chem. 34, 479-482. reading the manuscript. This investigation was supported in part by 9. Pande, S. V. & Mead, J. F. (1968) J. Biol. Chem. 243, 352- research grants from the Ministry of Education of Japan, the Mitsu- 361. bishi Foundation, the Foundation for the Promotion of Research on 10. Banis, R. J. & Tove, S. B. (1974) Biochim. Biophys. Acta 348, Medicinal Resources, the Japanese Foundation of Metabolism and 210-220. Diseases, and the Japanese Medical Association. S.P. is a recipient of 11. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. a Japanese Government Scholarship. (1951) J. Biol. Chem. 193,265-275. 12. Patterson, M. S. & Greene, R. C. (1965) Anal. Chem. 37, 854-857. 1. Lynen, F. (1959) J. Cell. Comp. Physiol. 54, Suppl. 1, 33-49. 13. Yamashita, S., Nakaya, N., Miki, Y. & Numa, S. (1975) Proc. 2. Kamiryo, T. & Numa, S. (1973) FEBS Lett. 38, 29-32. Nat. Acad. Sci. USA 72,600-603. 3. Numa, S. & Yamashita, S. (1974) Curr. Top. Cell. Regul. 8, 14. Lynen, F., Hopper-Kessel, I. & Eggerer, H. (1964) Biochem. 197-246. Z. 340, 95-124. 4. Jacobs, R. A., Sly, W. S. & Majerus, P. W. (1973) J. Biol. 15. Omura, S., Katagiri, M., Nakagawa, A., Sano, Y., Nomura, S. Chem. 248, 1268-1276. & Hata, T. (1967) J. Antibiot. Ser. A 20,349-354. 5. Kitajima, K., Tashiro, S. & Numa, S. (1975) Eur. J. Biochem. 16. Vance, D., Goldberg, I., Mitsuhashi, O., Bloch, K., Omura, S. 54,373-383. & Nomura, S. (1972) Blochem. Biophys. Res. Commun. 48, 6. Hosaka, K., Yamashita, S. & Numa, S. (1975) J. Biochem. 649-656. (Tokyo) 77,501-509. 17. Klein, K., Steinberg, R., Fiethen, B. & Overath, P. (1971) Eur. 7. Fink, G. R. (1970) in Methods in Enzymology, eds. Tabor, H. J. Biochem. 19,442450. & Tabor, C. W. (Academic Press, New York), Vol. 17A, pp. 18. Orme, T. W., McIntyre, J., Lynen, F., Kuhn, L. & Schweizer, 59-78. E. (1972) Eur. J. Biochem. 24,407-415. Downloaded by guest on September 27, 2021